Devices And Method For Enrichment And Alteration Of Cells And Other Particles

ABSTRACT

The invention features devices and methods for the deterministic separation of particles. Exemplary methods include the enrichment of a sample in a desired particle or the alteration of a desired particle in the device. The devices and methods are advantageously employed to enrich for rare cells, e.g., fetal cells, present in a sample, e.g., maternal blood and rare cell components, e.g., fetal cell nuclei. The invention further provides a method for preferentially lysing cells of interest in a sample, e.g., to extract clinical information from a cellular component, e.g., a nucleus, of the cells of interest. In general, the method employs differential lysis between the cells of interest and other cells (e.g., other nucleated cells) in the sample.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/965,128, filed on Apr. 27, 2018, which is a continuation of U.S.application Ser. No. 14/930,313, filed on Nov. 2, 2015 (now U.S. Pat.No. 9,956,562), which is a divisional of U.S. application Ser. No.13/232,781, filed on Sep. 14, 2011 (now U.S. Pat. No. 9,174,222), whichis a continuation of U.S. application Ser. No. 11/449,149, filed on Jun.8, 2006 (now U.S. Pat. No. 8,021,614), which is a continuation ofInternational Application No. PCT/US2006/012820, filed on Apr. 5, 2006,which further claims the benefit of U.S. Provisional Application No.60/668,415, filed on Apr. 5, 2005, and 60/704,067, filed on Jul. 29,2005. All of the aforementioned applications are incorporated herein byreference in their entireties.

BACKGROUND OF THE INVENTION

The invention relates to the fields of cell separation and fluidicdevices. Clinically or environmentally relevant information may often bepresent in a sample, but in quantities too low to detect. Thus, variousenrichment or amplification methods are often employed in order toincrease the detectability of such information.

For cells, different flow cytometry and cell sorting methods areavailable, but these techniques typically employ large and expensivepieces of equipment, which require large volumes of sample and skilledoperators. These cytometers and sorters use methods like electrostaticdeflection, centrifugation, fluorescence activated cell sorting (FACS),and magnetic activated cell sorting (MACS) to achieve cell separation.These methods often suffer from the inability to enrich a samplesufficiently to allow analysis of rare components of the sample.

Furthermore, such techniques may result in unacceptable losses of suchrare components, e.g., through inefficient separation or degradation ofthe components.

Thus, there is a need for new devices and methods for enriching samples.

SUMMARY OF THE INVENTION

In general, the invention features devices that contain one or morestructures that deterministically deflect particles, in a fluid, havinga hydrodynamic size above a critical size in a direction not parallel tothe average direction of flow of the fluid in the structure. Anexemplary structure includes an array of obstacles that form a networkof gaps, wherein a fluid passing through the gaps is divided unequallyinto a major flux and a minor flux so that the average direction of themajor flux is not parallel to the average direction of fluidic flow inthe channel, and the major flux from the first outer region is directedeither toward the second outer region or away from the second outerregion, wherein the particles are directed into the major flux. Thearray of obstacles preferably includes first and second rows displacedlaterally relative to one another so that fluid passing through a gap inthe first row is divided unequally into two gaps in the second row. Suchstructures may be arranged in series in a single channel, in parallel inthe same channel, e.g., a duplex configuration, in parallel in multiplechannels in a device, or combinations thereof. Each channel will have atleast one inlet and at least one outlet. A single inlet and outlet maybe employed for two or more structures in parallel, in the same ordifferent channels. Alternatively, each structure may have its own inletand outlet or a single structure may contain multiple inlets andoutlets, e.g., to introduce or collect two different fluidssimultaneously.

The invention further features methods of enriching and altering samplesemploying a device of the invention.

In preferred embodiments, the devices of the invention includemicrofluidic channels. In other preferred embodiments, the devices ofthe invention are configured to separate blood components, e.g., redblood cells, white blood cells, or platelets from whole blood, rarecells such as nucleated red blood cells from maternal blood, and stemcells, pathogenic or parasitic organisms, or host or graft immune cellsfrom blood. The methods may also be employed to separate all bloodcells, or portions thereof, from plasma, or all particles in a samplesuch as cellular components or intracellular parasites, or subsetsthereof, from the suspending fluid. Other particles that may beseparated in devices of the invention are described herein.

The invention further provides methods for preferentially lysing cellsof interest in a sample, e.g., to extract clinical information from acellular component, e.g., a nucleus or nucleic acid, of the cells ofinterest, e.g., nucleated fetal red blood cells. In general, the methodemploys differential lysis between the cells of interest and other cells(e.g., other nucleated cells) in the sample. In certain embodiments,preferential lysis results in lysis of at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95%, or 99% of cells of interest, e.g., red bloodcells or fetal nucleated red blood cells, and lysis of less than 50%,40%, 30%, 20%, 10%, 5%, or 1% of undesired cells, e.g. maternal whiteblood cells or maternal nucleated red blood cells.

By “gap” is meant an opening through which fluids and/or particles mayflow. For example, a gap may be a capillary, a space between twoobstacles wherein fluids may flow, or a hydrophilic pattern on anotherwise hydrophobic surface wherein aqueous fluids are confined. In apreferred embodiment of the invention, the network of gaps is defined byan array of obstacles. In this embodiment, the gaps are the spacesbetween adjacent obstacles. In a preferred embodiment, the network ofgaps is constructed with an array of obstacles on the surface of asubstrate.

By “obstacle” is meant an impediment to flow in a channel, e.g., aprotrusion from one surface. For example, an obstacle may refer to apost outstanding on a base substrate or a hydrophobic barrier foraqueous fluids. In some embodiments, the obstacle may be partiallypermeable. For example, an obstacle may be a post made of porousmaterial, wherein the pores allow penetration of an aqueous componentbut are too small for the particles being separated to enter.

By “hydrodynamic size” is meant the effective size of a particle wheninteracting with a flow, posts, and other particles. It is used as ageneral term for particle volume, shape, and deformability in the flow.

By “flow-extracting boundary” is meant a boundary designed to removefluid from an array.

By “flow-feeding boundary” is meant a boundary designed to add fluid toan array.

By “swelling reagent” is meant a reagent that increases the hydrodynamicradius of a particle. Swelling reagents may act by increasing thevolume, reducing the deformability, or changing the shape of a particle.

By “shrinking reagent” is meant a reagent that decreases thehydrodynamic radius of a particle. Shrinking reagents may act bydecreasing the volume, increasing the deformability, or changing theshape of a particle.

By “labeling reagent” is meant a reagent that is capable of binding toor otherwise being localized with a particle and being detected, e.g.,through shape, morphology, color, fluorescence, luminescence,phosphorescence, absorbance, magnetic properties, or radioactiveemission.

By “channel” is meant a gap through which fluid may flow. A channel maybe a capillary, a conduit, or a strip of hydrophilic pattern on anotherwise hydrophobic surface wherein aqueous fluids are confined.

By “microfluidic” is meant having at least one dimension of less than 1mm.

By “enriched sample” is meant a sample containing cells or otherparticles that has been processed to increase the relative population ofcells or particles of interest relative to other components typicallypresent in a sample. For example, samples may be enriched by increasingthe relative population of particles of interest by at least 10%, 25%,50%, 75%, 100% or by a factor of at least 1000, 10,000, 100,000, or1,000,000.

By “intracellular activation” is meant activation of second messengerpathways, leading to transcription factor activation, or activation ofkinases or other metabolic pathways. Intracellular activation throughmodulation of external cell membrane antigens can also lead to changesin receptor trafficking.

By “cellular sample” is meant a sample containing cells or componentsthereof. Such samples include naturally occurring fluids (e.g., blood,lymph, cerebrospinal fluid, urine, cervical lavage, and water samples)and fluids into which cells have been introduced (e.g., culture media,and liquefied tissue samples). The term also includes a lysate.

By “biological sample” is meant any same of biological origin orcontaining, or potentially containing, biological particles. Preferredbiological samples are cellular samples.

By “biological particle” is meant any species of biological origin thatis insoluble in aqueous media. Examples include cells, particulate cellcomponents, viruses, and complexes including proteins, lipids, nucleicacids, and carbohydrates.

By “component” of a cell (or “cellular component”) is meant anycomponent of a cell that may be at least partially isolated upon lysisof the cell. Cellular components may be organelles (e.g., nuclei,peri-nuclear compartments, nuclear membranes, mitochondria,chloroplasts, or cell membranes), polymers or molecular complexes (e.g.,lipids, polysaccharides, proteins (membrane, trans-membrane, orcytosolic), nucleic acids (native, therapeutic, or pathogenic), viralparticles, or ribosomes), intracellular parasites or pathogens, or othermolecules (e.g., hormones, ions, cofactors, or drugs).

By “blood component” is meant any component of whole blood, includinghost red blood cells, white blood cells, and platelets. Blood componentsalso include the components of plasma, e.g., proteins, lipids, nucleicacids, and carbohydrates, and any other cells that may be present inblood, e.g., because of current or past pregnancy, organ transplant, orinfection.

By “counterpart” is meant a cellular component, which although differentat the detail level (e.g., sequence) is of the same class. Examples arenuclei, mitochondria, mRNA, and ribosomes from different cell types,e.g., fetal red blood cells and maternal white blood cells.

By “preferential lysis” is meant lysing a cell of interest to a greaterextent than undesired cells on the time scale of the lysis. Undesiredcells typically contain the same cellular component found in the cellsof interest or a counterpart thereof or cellular components that damagethe contents of cells of interest. Preferential lysis may result inlysis of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or99% of cells of interest, e.g., while lysing less than 50%, 40%, 30%,20%, 10%, 5%, or 1% of undesired cells. Preferential lysis may alsoresult in a ratio of lysis of cells of interest to undesired cells.

Other features and advantages will be apparent from the followingdescription and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.

Copies of this patent with color drawings will be provided by the Patentand Trademark Office upon request and payment of the necessary fee.

FIGS. 1A-1E are schematic depictions of an array that separated cellsbased on deterministic lateral displacement: (A) illustrates the lateraldisplacement of subsequent rows; (B) illustrates how fluid flowingthrough a gap is divide unequally around obstacles in subsequent rows;(C) illustrates how a particle with a hydrodynamic size above thecritical size is displaced laterally in the device; (D) illustrates anarray of cylindrical obstacles; and (E) illustrates an array ofelliptical obstacles.

FIG. 2 is a schematic description illustrating the unequal division ofthe flux through a gap around obstacles in subsequent rows.

FIG. 3 is a schematic depiction of how the critical size depends on theflow profile, which is parabolic in this example.

FIG. 4 is an illustration of how shape affects the movement of particlesthrough a device.

FIG. 5 is an illustration of how deformability affects the movement ofparticles through a device.

FIG. 6 is a schematic depiction of deterministic lateral displacement.Particles having a hydrodynamic size above the critical size move to theedge of the array, while particles having a hydrodynamic size below thecritical size pass through the device without lateral displacement.

FIG. 7 is a schematic depiction of a three-stage device.

FIG. 8 is a schematic depiction of the maximum size and cut-off size(i.e., critical size) for the device of FIG. 7.

FIG. 9 is a schematic depiction of a bypass channel.

FIG. 10 is a schematic depiction of a bypass channel.

FIG. 11 is a schematic depiction of a three-stage device having a commonbypass channel.

FIG. 12 is a schematic depiction of a three-stage, duplex device havinga common bypass channel.

FIG. 13 is a schematic depiction of a three-stage device having a commonbypass channel, where the flow through the device is substantiallyconstant.

FIG. 14 is a schematic depiction of a three-stage, duplex device havinga common bypass channel, where the flow through the device issubstantially constant.

FIG. 15 is a schematic depiction of a three-stage device having a commonbypass channel, where the fluidic resistance in the bypass channel andthe adjacent stage are substantially constant.

FIG. 16 is a schematic depiction of a three-stage, duplex device havinga common bypass channel, where the fluidic resistance in the bypasschannel and the adjacent stage are substantially constant.

FIG. 17 is a schematic depiction of a three-stage device having two,separate bypass channels.

FIG. 18 is a schematic depiction of a three-stage device having two,separate bypass channels, which are in arbitrary configuration.

FIG. 19 is a schematic depiction of a three-stage, duplex device havingthree, separate bypass channels.

FIG. 20 is a schematic depiction of a three-stage device having two,separate bypass channels, wherein the flow through each stage issubstantially constant.

FIG. 21 is a schematic depiction of a three-stage, duplex device havingthree, separate bypass channels, wherein the flow through each stage issubstantially constant.

FIG. 22 is a schematic depiction of a flow-extracting boundary.

FIG. 23 is a schematic depiction of a flow-feeding boundary.

FIG. 24 is a schematic depiction of a flow-feeding boundary, including abypass channel.

FIG. 25 is a schematic depiction of two flow-feeding boundaries flankinga central bypass channel.

FIG. 26 is a schematic depiction of a device having four channels thatact as on-chip flow resistors.

FIGS. 27 and 28 are schematic depictions of the effect of on-chipresistors on the relative width of two fluids flowing in a device.

FIG. 29 is a schematic depiction of a duplex device having a commoninlet for the two outer regions.

FIG. 30A is a schematic depiction of a multiple arrays on a device.

FIG. 30B is a schematic depiction of multiple arrays with common inletsand product outlets on a device.

FIG. 31 is a schematic depiction of a multi-stage device with a smallfootprint.

FIG. 32 is a schematic depiction of blood passing through a device.

FIG. 33 is a graph illustrating the hydrodynamic size distribution ofblood cells.

FIGS. 34A-34D are schematic depictions of moving a particle from asample to a buffer in a single stage (A), three-stage (B), duplex (C),or three-stage duplex (D) device.

FIG. 35A is a schematic depiction of a two-stage device employed to movea particle from blood to a buffer to produce three products. FIG. 35B isa schematic graph of the maximum size and cut off size of the twostages. FIG. 35C is a schematic graph of the composition of the threeproducts.

FIG. 36 is a schematic depiction of a two-stage device for alteration,where each stage has a bypass channel.

FIG. 37 is a schematic depiction of the use of fluidic channels toconnect two stages in a device.

FIG. 38 is a schematic depiction of the use of fluidic channels toconnect two stages in a device, wherein the two stages are configured asa small footprint array.

FIG. 39A is a schematic depiction of a two-stage device having a bypasschannel that accepts output from both stages. FIG. 39B is a schematicgraph of the range of product sizes achievable with this device.

FIG. 40 is a schematic depiction of a two-stage device for alterationhaving bypass channels that flank each stage and empty into the sameoutlet.

FIG. 41 is a schematic depiction of a device for the sequential movementand alteration of particles.

FIG. 42A is a photograph of a device of the invention. FIGS. 42B-42E aredepictions the mask used to fabricate a device of the invention. FIG.42F is a series of photographs of the device containing blood andbuffer.

FIGS. 43A-43F are typical histograms generated by the hematologyanalyzer from a blood sample and the waste (buffer, plasma, red bloodcells, and platelets) and product (buffer and nucleated cells) fractionsgenerated by the device of FIG. 42.

FIGS. 44A-44D are depictions the mask used to fabricate a device of theinvention.

FIGS. 45A-45D are depictions the mask used to fabricate a device of theinvention.

FIG. 46A is a micrograph of a sample enriched in fetal red blood cells.FIG. 46B is a micrograph of maternal red blood cell waste.

FIG. 47 is a series of micrographs showing the positive identificationof male fetal cells (Blue=nucleus, Red=X chromosome, Green=Ychromosome).

FIG. 48 is a series of micrographs showing the positive identificationof sex and trisomy 21.

FIGS. 49A-49D are depictions the mask used to fabricate a device of theinvention.

FIGS. 50A-50G are electron micrographs of the device of FIG. 49.

FIGS. 51A-51D are depictions the mask used to fabricate a device of theinvention.

FIGS. 52A-52F are electron micrographs of the device of FIG. 51.

FIGS. 53A-53F are electron micrographs of the device of FIG. 45.

FIGS. 54A-54D are depictions the mask used to fabricate a device of theinvention.

FIGS. 55A-55S are electron micrographs of the device of FIG. 54.

FIGS. 56A-56C are electron micrographs of the device of FIG. 44.

FIG. 57 is a flowchart describing the isolation of fetal red blood cellnuclei.

FIG. 58 is a schematic graph of the course of lysis of cells in amaternal blood sample.

FIG. 59 is a schematic diagram of a microfluidic method to enrich thecells of interest and preferentially lyse the cells of interest in theenriched sample. The sample is first enriched by size-based direction ofcells of interest into a preferred channel, and the cells of interestare then selectively lysed by controlling their residence time in alysis solution.

FIG. 60 is a schematic diagram of a microfluidic method of size-basedisolation of the nuclei of the lysed cells of interest from non-lysedwhole cells of non-interest. The cells of non-interest are directed intothe waste, while the nuclei are retained in the desired product streams.

FIG. 61 is a flowchart describing an alternate method for the separationof fetal nuclei from maternal white blood cells.

FIG. 62 is a schematic diagram of a device of the invention employing asubstantially constant gap width and flow-feeding and flow-extractingboundaries.

FIG. 63a is a schematic depiction of a manifold of the invention. FIG.63b is a photograph of a manifold of the invention.

FIG. 64 is a graph of the percentage of viable cells as a function ofexposure to a hypotonic lysis solution.

FIG. 65 is a graph of hemolysis of whole blood as a function of time ina lysis buffer.

FIG. 66 is a table that illustrates the nuclei recovery after Cytospinusing Carney's fix solution total cell lysis procedure as describedherein.

FIG. 67 is a flowchart detailing various options for lysis of cells andnuclei.

DETAILED DESCRIPTION OF THE INVENTION Device

In general, the devices include one or more arrays of obstacles thatallow deterministic lateral displacement of components of fluids. Priorart devices that differ from those the present invention, but which,like those of the invention, employ obstacles for this purpose aredescribed, e.g., in Huang et al. Science 304, 987-990 (2004) and U.S.Publication No. 20040144651. The devices of the invention for separatingparticles according to size employ an array of a network of gaps,wherein a fluid passing through a gap is divided unequally intosubsequent gaps. The array includes a network of gaps arranged such thatfluid passing through a gap is divided unequally, even though the gapsmay be identical in dimensions.

The device uses a flow that carries cells to be separated through thearray of gaps. The flow is aligned at a small angle (flow angle) withrespect to a line-of-sight of the array. Cells having a hydrodynamicsize larger than a critical size migrate along the line-of-sight in thearray, whereas those having a hydrodynamic size smaller than thecritical size follow the flow in a different direction. Flow in thedevice occurs under laminar flow conditions.

The critical size is a function of several design parameters. Withreference to the obstacle array in FIG. 1, each row of posts is shiftedhorizontally with respect to the previous row by Δλ, where λ is thecenter-to-center distance between the posts (FIG. 1A). The parameterΔλ/λ (the “bifurcation ratio,” ε) determines the ratio of flowbifurcated to the left of the next post. In FIG. 1, ε is 1/3, for theconvenience of illustration. In general, if the flux through a gapbetween two posts is ϕ, the minor flux is εϕ, and the major flux is(1-εϕ□) (FIG. 2). In this example, the flux through a gap is dividedessentially into thirds (FIG. 1B). While each of the three fluxesthrough a gap weaves around the array of posts, the average direction ofeach flux is in the overall direction of flow. FIG. 1C illustrates themovement of a particles sized above the critical size through the array.Such particles move with the major flux, being transferred sequentiallyto the major flux passing through each gap.

Referring to FIG. 2, the critical size is approximately 2R_(critical),where R_(critical) is the distance between the stagnant flow line andthe post. If the center of mass of a particle, e.g., a cell, falls atleast R_(critical) away from the post, the particle would follow themajor flux and move along the line-of-sight of the array. If the centerof mass of a particle falls within R_(critical) of the post, it followsthe minor flux in a different direction. R_(critical) can be determinedif the flow profile across the gap is known (FIG. 3); it is thethickness of the layer of fluids that would make up the minor flux. Fora given gap size, d, R_(critical) can be tailored based on thebifurcation ratio, ε. In general, the smaller ε, the smallerR_(critical).

In an array for deterministic lateral displacement, particles ofdifferent shapes behave as if they have different sizes (FIG. 4). Forexample, lymphocytes are spheres of ˜5 μm diameter, and erythrocytes arebiconcave disks of ˜7 μm diameter, and ˜1.5 μm thick. The long axis oferythrocytes (diameter) is larger than that of the lymphocytes, but theshort axis (thickness) is smaller. If erythrocytes align their long axesto a flow when driven through an array of posts by the flow, theirhydrodynamic size is effectively their thickness (˜1.5 μm), which issmaller than lymphocytes. When an erythrocyte is driven through an arrayof posts by a hydrodynamic flow, it tends to align its long axis to theflow and behave like a ˜1.5 μm-wide particle, which is effectively“smaller” than lymphocytes. The method and device may therefore separatecells according to their shapes, although the volumes of the cells couldbe the same. In addition, particles having different deformabilitybehave as if they have different sizes (FIG. 5). For example, twoparticles having the undeformed shape may be separated by deterministiclateral displacement, as the cell with the greater deformability maydeform when it comes into contact with an obstacle in the array andchange shape. Thus, separation in the device may be achieved based onany parameter that affects hydrodynamic size including the physicaldimensions, the shape, and the deformability of the particle.

Referring to FIGS. 6 and 7, feeding a mixture of particles, e.g., cells,of different hydrodynamic sizes from the top of the array and collectingthe particles at the bottom, as shown schematically, produces twoproducts, the output containing cells larger than the critical size,2R_(critical), and waste containing cells smaller than the criticalsize. Although labeled “waste” in FIG. 7, particles below the criticalsize may be collected while the particles above the critical size arediscarded. Both types of outputs may also be desirably collected, e.g.,when fractionating a sample into two or more sub-samples. Cells largerthan the gap size will get trapped inside the array. Therefore, an arrayhas a working size range. Cells have to be larger than a critical size(2R_(critical)) and smaller than a maximum pass-through size (array gapsize) to be directed into the major flux.

Uses of Devices of the Invention

The invention features improved devices for the separation of particles,including bacteria, viruses, fungi, cells, cellular components, viruses,nucleic acids, proteins, and protein complexes, according to size. Thedevices may be used to effect various manipulations on particles in asample. Such manipulations include enrichment or concentration of aparticle, including size based fractionization, or alteration of theparticle itself or the fluid carrying the particle. Preferably, thedevices are employed to enrich rare particles from a heterogeneousmixture or to alter a rare particle, e.g., by exchanging the liquid inthe suspension or by contacting a particle with a reagent. Such devicesallow for a high degree of enrichment with limited stress on cells,e.g., reduced mechanical lysis or intracellular activation of cells.

Although primarily described in terms of cells, the devices of theinvention may be employed with any other particles whose size allows forseparation in a device of the invention.

Array Design

Single-stage array. In one embodiment, a single stage contains an arrayof obstacles, e.g., cylindrical posts (FIG. 1D). In certain embodiments,the array has a maximum pass-through size that is several times largerthan the critical size, e.g., when separating white blood cells from redblood cells. This result may be achieved using a combination of a largegap size d and a small bifurcation ratio ε. In preferred embodiments,the £ is at most 1/2, e.g., at most 1/3, 1/10, 1/30, 1/100, 1/300, or1/1000. In such embodiments, the obstacle shape may affect the flowprofile in the gap; however, the obstacles can be compressed in the flowdirection, in order to make the array short (FIG. 1E). Single stagearrays may include bypass channels as described herein.

Multiple-stage arrays. In another embodiment, multiple stages areemployed to separate particles over a wide range of sizes. An exemplarydevice is shown in FIG. 7. The device shown has three stages, but anynumber of stages may be employed. Typically, the cut-off size (i.e.critical size) in the first stage is larger than the cut-off in thesecond stage, and the first stage cut-off size is smaller than themaximum pass-through size of the second stage (FIG. 8). The same is truefor the following stages. The first stage will deflect (and remove)particles, e.g., that would cause clogging in the second stage, beforethey reach the second stage. Similarly, the second stage will deflect(and remove) particles that would cause clogging in the third stage,before they reach the third stage. In general an array can have as manystages as desired.

As described, in a multiple-stage array, large particles, e.g., cells,that could cause clogging downstream are deflected first, and thesedeflected particles need to bypass the downstream stages to avoidclogging. Thus, devices of the invention may include bypass channelsthat remove output from an array. Although described here in terms ofremoving particles above the critical size, bypass channels may also beemployed to remove output from any portion of the array.

Different designs for bypass channels are as follows.

Single bypass channels. In this design, all stages share one bypasschannel, or there is only one stage. The physical boundary of the bypasschannel may be defined by the array boundary on one side and a sidewallon the other side (FIGS. 9-11). Single bypass channels may also beemployed with duplex arrays such that a central bypass channel separatesthe two arrays (i.e., two outer regions) (FIG. 12).

Single bypass channels may also be designed, in conjunction with anarray to maintain constant flux through a device (FIG. 13). The bypasschannel has varying width designed to maintain constant flux through allthe stages, so that the flow in the channel does not interfere with theflow in the arrays. Such a design may also be employed with an arrayduplex (FIG. 14). Single bypass channels may also be designed inconjunction with the array in order to maintain substantially constantfluidic resistance through all stages (FIG. 15). Such a design may alsobe employed with an array duplex (FIG. 16.)

Multiple bypass channels. In this design (FIG. 17), each stage has itsown bypass channel, and the channels are separated from each other bysidewalls, e.g., to prevent the mixing of the contents of differentchannels. Large particles, e.g., cells are deflected into the major fluxto the lower right corner of the first stage and then into in a bypasschannel (bypass channel 1 in FIG. 17). Smaller cells that would notcause clogging in the second stage proceed to the second stage, andcells above the critical size of the second stage are deflected to thelower right corner of the second stage and into in another bypasschannel (bypass channel 2 in FIG. 17). This design may be repeated foras many stages as desired. In this embodiment, the bypass channels arenot fluidically connected, allowing for separate collection and othermanipulations. The bypass channels do not need to be straight or bephysically parallel to each other (FIG. 18). Multiple bypass channelsmay also be employed with duplex arrays (FIG. 19).

Multiple bypass channels may be designed, in conjunction with an arrayto maintain constant flux through a device (FIG. 20). In this example,bypass channels are designed to remove an amount of flow so the flow inthe array is not perturbed, i.e., substantially constant. Such a designmay also be employed with an array duplex (FIG. 21). In this design, thecenter bypass channel may be shared between the two arrays in theduplex.

Optimal Boundary Design. If the array were infinitely large, the flowdistribution would be the same at every gap. The flux ϕ going through agap would be the same, and the minor flux would be εϕ for every gap. Inpractice, the boundaries of the array perturb this infinite flowpattern. Portions of the boundaries of arrays may be designed togenerate the flow pattern of an infinite array. Boundaries may beflow-feeding, i.e., the boundary injects fluid into the array, orflow-extracting, i.e., the boundary extracts fluid from the array.

A preferred flow-extracting boundary widens gradually to extract εϕ(represented by arrows in FIG. 22) from each gap at the boundary (d=24μm, ε=1/60). For example, the distance between the array and thesidewall gradually increases to allow for the addition of εϕ to theboundary from each gap along that boundary. The flow pattern inside thisarray is not affected by the bypass channel because of the boundarydesign.

A preferred flow-feeding boundary narrows gradually to feed exactly εϕ(represented by arrows in FIG. 23) into each gap at the boundary (d=24μm, ε=1/60). For example, the distance between the array and thesidewall gradually decreases to allow for the addition of εϕ to each gapalong the boundary from that boundary. Again, the flow pattern insidethis array is not affected by the bypass channel because of the boundarydesign.

A flow-feeding boundary may also be as wide as or wider than the gaps ofan array (FIG. 24) (d=24 μm, ε=1/60). A wide boundary may be desired ifthe boundary serves as a bypass channel, e.g., to allow for collectionof particles. A boundary may be employed that uses part of its entireflow to feed the array and feeds εϕ into each gap at the boundary(represented by arrows in FIG. 24).

FIG. 25 shows a single bypass channel in a duplex array (ε=1/10, d=8μm). The bypass channel includes two flow-feeding boundaries. The fluxacross the dashed line 1 in the bypass channel is Φ_(bypass). A flow ϕjoins Φ_(bypass) from a gap to the left of the dashed line. The shapesof the obstacles at the boundaries are adjusted so that the flows goinginto the arrays are s at each gap at the boundaries. The flux at dashedline 2 is again Φ_(bypass).

Device Design

On-Chip Flow Resistor for Defining and Stabilizing Flow

Devices of the invention may also employ fluidic resistors to define andstabilize flows within an array and to also define the flows collectedfrom the array. FIG. 26 shows a schematic of planar device; a sample,e.g., blood, inlet channel, a buffer inlet channel, a waste outletchannel, and a product outlet channel are each connected to an array.The inlets and outlets act as flow resistors. FIG. 26 also shows thecorresponding fluidic resistances of these different device components.

Flow Definition within the Array

FIGS. 27 and 28 show the currents and corresponding widths of the sampleand buffer flows within the array when the device has a constant depthand is operated with a given pressure drop. The flow is determined bythe pressure drop divided by the resistance. In this particular device,blood and buffer are equivalent, and this determines equivalent widthsof the blood and buffer streams in the array.

Definition of Collection Fraction

By controlling the relative resistance of the product and waste outletchannels, one can modulate the collection tolerance for each fraction.For example, in this particular set of schematics, when R_(product) isgreater than R_(waste), a more concentrated product fraction will resultat the expense of a potentially increased loss to and dilution of wastefraction. Conversely, when R_(product) is less than R_(waste), a moredilute and higher yield product fraction will be collected at theexpense of potential contamination from the waste stream.

Flow Stabilization

Each of the inlet and outlet channels can be designed so that thepressure drops across the channels are appreciable to or greater thanthe fluctuations of the overall driving pressure. In typical cases, theinlet and outlet pressure drops are 0.001 to 0.99 times the drivingpressure.

Multiplexed Arrays

The invention features multiplexed arrays. Putting multiple arrays onone device increases sample-processing throughput and allows forparallel processing of multiple samples or portions of the sample fordifferent fractions or manipulations. Multiplexing is further desirablefor preparative devices. The simplest multiplex device includes twodevices attached in series, i.e., a cascade. For example, the outputfrom the major flux of one device may be coupled to the input of asecond device. Alternatively, the output from the minor flux of onedevice may be coupled to the input of the second device.

Duplexing. Two arrays can be disposed side-by-side, e.g., as minorimages (FIG. 29). In such an arrangement, the critical size of the twoarrays may be the same or different. Moreover, the arrays may bearranged so that the major flux flows to the boundary of the two arrays,to the edge of each array, or a combination thereof. Such a duplexedarray may also contain a central bypass channel disposed between thearrays, e.g., to collect particles above the critical size or to alterthe sample, e.g., through buffer exchange, reaction, or labeling.

Multiplexing on a device. In addition to forming a duplex, two or morearrays that have separated inputs may be disposed on the same device(FIG. 30A). Such an arrangement could be employed for multiple samples,or the plurality of arrays may be connected to the same inlet forparallel processing of the same sample. In parallel processing of thesame sample, the outlets may or may not be fluidically connected. Forexample, when the plurality of arrays has the same critical size, theoutlets may be connected for high throughput sample processing. Inanother example, the arrays may not all have the same critical size orthe particles in the arrays may not all be treated in the same manner,and the outlets may not be fluidically connected.

Multiplexing may also be achieved by placing a plurality of duplexarrays on a single device (FIG. 30B). A plurality of arrays, duplex orsingle, may be placed in any possible three-dimensional relationship toone another.

Devices of the invention also feature a small-footprint. Reducing thefootprint of an array can lower cost, and reduce the number ofcollisions with obstacles to eliminate any potential mechanical damageor other effects to particles. The length of a multiple stage array canbe reduced if the boundaries between stages are not perpendicular to thedirection of flow. The length reduction becomes significant as thenumber of stages increases. FIG. 31 shows a small-footprint three-stagearray.

Additional Components

In addition to an array of gaps, devices of the invention may includeadditional elements, e.g., for isolating, collection, manipulation, ordetection. Such elements are known in the art. Arrays may also beemployed on a device having components for other types of separation,including affinity, magnetic, electrophoretic, centrifugal, anddielectrophoretic separation. Devices of the invention may also beemployed with a component for two-dimensional imaging of the output fromthe device, e.g., an array of wells or a planar surface. Preferably,arrays of gaps as described herein are employed in conjunction with anaffinity enrichment.

The invention may also be employed in conjunction with other enrichmentdevices, either on the same device or in different devices. Otherenrichment techniques are described, e.g., in International PublicationNos. 2004/029221 and 2004/113877, U.S. Pat. No. 6,692,952, U.S.Application Publications 2005/0282293 and 2005/0266433, and U.S.Application No. 60/668,415, each of which is incorporated by reference.

Methods of Fabrication

Devices of the invention may be fabricated using techniques well knownin the art. The choice of fabrication technique will depend on thematerial used for the device and the size of the array. Exemplarymaterials for fabricating the devices of the invention include glass,silicon, steel, nickel, poly(methylmethacrylate) (PMMA), polycarbonate,polystyrene, polyethylene, polyolefins, silicones (e.g.,poly(dimethylsiloxane)), and combinations thereof. Other materials areknown in the art. For example, deep Reactive Ion Etching (DRIE) is usedto fabricate silicon-based devices with small gaps, small obstacles andlarge aspect ratios (ratio of obstacle height to lateral dimension).Thermoforming (embossing, injection molding) of plastic devices can alsobe used, e.g., when the smallest lateral feature is 20 microns and theaspect ratio of these features is less than 3 Additional methods includephotolithography (e.g., stereolithography or x-ray photolithography),molding, embossing, silicon micromachining, wet or dry chemical etching,milling, diamond cutting, Lithographie Galvanoformung and Abformung(LIGA), and electroplating. For example, for glass, traditional siliconfabrication techniques of photolithography followed by wet (KOH) or dryetching (reactive ion etching with fluorine or other reactive gas) canbe employed. Techniques such as laser micromachining can be adopted forplastic materials with high photon absorption efficiency. This techniqueis suitable for lower throughput fabrication because of the serialnature of the process. For mass-produced plastic devices, thermoplasticinjection molding, and compression molding may be suitable. Conventionalthermoplastic injection molding used for mass-fabrication of compactdiscs (which preserves fidelity of features in sub-microns) may also beemployed to fabricate the devices of the invention. For example, thedevice features are replicated on a glass master by conventionalphotolithography. The glass master is electroformed to yield a tough,thermal shock resistant, thermally conductive, hard mold. This moldserves as the master template for injection molding or compressionmolding the features into a plastic device. Depending on the plasticmaterial used to fabricate the devices and the requirements on opticalquality and throughput of the finished product, compression molding orinjection molding may be chosen as the method of manufacture.Compression molding (also called hot embossing or relief imprinting) hasthe advantages of being compatible with high-molecular weight polymers,which are excellent for small structures, but is difficult to use inreplicating high aspect ratio structures and has longer cycle times.Injection molding works well for high-aspect ratio structures but ismost suitable for low molecular weight polymers.

A device may be fabricated in one or more pieces that are thenassembled. Layers of a device may be bonded together by clamps,adhesives, heat, anodic bonding, or reactions between surface groups(e.g., wafer bonding). Alternatively, a device with channels in morethan one plane may be fabricated as a single piece, e.g., usingstereolithography or other three-dimensional fabrication techniques.

To reduce non-specific adsorption of cells or compounds, e.g., releasedby lysed cells or found in biological samples, onto the channel walls,one or more channel walls may be chemically modified to be non-adherentor repulsive. The walls may be coated with a thin film coating (e.g., amonolayer) of commercial non-stick reagents, such as those used to formhydrogels. Additional examples chemical species that may be used tomodify the channel walls include oligoethylene glycols, fluorinatedpolymers, organosilanes, thiols, poly-ethylene glycol, hyaluronic acid,bovine serum albumin, poly-vinyl alcohol, mucin, poly-HEMA,methacrylated PEG, and agarose. Charged polymers may also be employed torepel oppositely charged species. The type of chemical species used forrepulsion and the method of attachment to the channel walls will dependon the nature of the species being repelled and the nature of the wallsand the species being attached. Such surface modification techniques arewell known in the art. The walls may be functionalized before or afterthe device is assembled. The channel walls may also be coated in orderto capture materials in the sample, e.g., membrane fragments orproteins.

Methods of Operation

Devices of the invention may be employed in any application where theproduction of a sample enriched in particles above or below a criticalsize is desired. A preferred use of the device is in produced samplesenriched in cells, e.g., rare cells. Once an enriched sample isproduced, it may be collected for analysis or otherwise manipulated,e.g., through further enrichment.

The method of the invention uses a flow that carries cells to beseparated through the array of gaps. The flow is aligned at a smallangle (flow angle) with respect to a line-of-sight of the array. Cellshaving a hydrodynamic size larger than a critical size migrate along theline-of-sight in the array, whereas those having a hydrodynamic sizesmaller than the critical size follow the flow in a different direction.Flow in the device occurs under laminar flow conditions.

The method of the invention may be employed with concentrated samples,e.g., where particles are touching, hydrodynamically interacting witheach other, or exerting an effect on the flow distribution aroundanother particle. For example, the method can separate white blood cellsfrom red blood cells in whole blood from a human donor. Human bloodtypically contains ˜45% of cells by volume. Cells are in physicalcontact and/or coupled to each other hydrodynamically when they flowthrough the array. FIG. 32 shows schematically that cells are denselypacked inside an array and could physically interact with each other.

Enrichment

In one embodiment, the methods of the invention are employed to producea sample enriched in particles of a desired hydrodynamic size.Applications of such enrichment include concentrating particles, e.g.,rare cells, and size fractionization, e.g., size filtering (selectingcells in a particular range of sizes). The methods may also be used toenrich components of cells, e.g., nuclei. Nuclei or other cellularcomponents may be produced by manipulation of the sample, e.g., lysis asdescribed herein, or be naturally present in the sample, e.g., viaapoptosis or necrosis. Desirably, the methods of the invention retain atleast 1%, 10%, 30%, 50%, 75%, 80%, 90%, 95%, 98%, or 99% of the desiredparticles compared to the initial mixture, while potentially enrichingthe desired particles by a factor of at least 1, 10, 100, 1000, 10,000,100,000, or even 1,000,000 relative to one or more non-desiredparticles. The enrichment may also result in a dilution of the separatedparticles compared to the original sample, although the concentration ofthe separated particles relative to other particles in the sample hasincreased. Preferably, the dilution is at most 90%, e.g., at most 75%,50%, 33%, 25%, 10%, or 1%.

In a preferred embodiment, the method produces a sample enriched in rareparticles, e.g., cells. In general, a rare particle is a particle thatis present as less than 10% of a sample. Exemplary rare particlesinclude, depending on the sample, fetal cells, nucleated red blood cells(e.g., fetal or maternal), stem cells (e.g., undifferentiated), cancercells, immune system cells (host or graft), epithelial cells, connectivetissue cells, bacteria, fungi, viruses, parasites, and pathogens (e.g.,bacterial or protozoan). Such rare particles may be isolated fromsamples including bodily fluids, e.g., blood, or environmental sources,e.g., pathogens in water samples. Fetal cells, e.g., nucleated RBCs, maybe enriched from maternal peripheral blood, e.g., for the purpose ofdetermining sex and identifying aneuploidies or genetic characteristics,e.g., mutations, in the developing fetus. Cancer cells may also beenriched from peripheral blood for the purpose of diagnosis andmonitoring therapeutic progress. Bodily fluids or environmental samplesmay also be screened for pathogens or parasites, e.g., for coliformbacteria, blood borne illnesses such as sepsis, or bacterial or viralmeningitis. Rare cells also include cells from one organism present inanother organism, e.g., an in cells from a transplanted organ.

In addition to enrichment of rare particles, the methods of theinvention may be employed for preparative applications. An exemplarypreparative application includes generation of cell packs from blood.The methods of the invention may be configured to produce fractionsenriched in platelets, red blood cells, and white cells. By usingmultiplexed devices or multistage devices, all three cellular fractionsmay be produced in parallel or in series from the same sample. In otherembodiments, the method may be employed to separate nucleated fromenucleated cells, e.g., from cord blood sources.

Using the methods of the invention is advantageous in situations wherethe particles being enriched are subject to damage or other degradation.As described herein, devices of the invention may be designed to enrichcells with a minimum number of collisions between the cells andobstacles. This minimization reduces mechanical damage to cells and alsoprevents intracellular activation of cells caused by the collisions.This gentle handling of the cells preserves the limited number of rarecells in a sample, prevents rupture of cells leading to contamination ordegradation by intracellular components, and prevents maturation oractivation of cells, e.g., stem cells or platelets. In preferredembodiments, cells are enriched such that fewer than 30%, 10%, 5%, 1%,0.1%, or even 0.01% are activated or mechanically lysed.

FIG. 33 shows a typical size distribution of cells in human peripheralblood. The white blood cells range from ˜4 μm to ˜18 μm, whereas the redblood cells are ˜1.5 μm (short axis). An array designed to separatewhite blood cells from red blood cells typically has a cut-off size(i.e., critical size) of 2 to 4 μm and a maximum pass-through size ofgreater than 18 μm.

In an alternative embodiment, the device would function as a detectorfor abnormalities in red blood cells. The deterministic principle ofsorting enables a predictive outcome of the percentage of enucleatedcells deflected in the device. In a disease state, such as malarialinfection or sickle cell anemia, the distortion in shape and flexibilityof the red cells would significantly change the percentage of cellsdeflected. This change can be monitored as a first level sentry to alertto the potential of a diseased physiology to be followed by microscopyexamination of shape and size of red cells to assign the disease. Themethod is also generally applicable monitoring for any change inflexibility of particles in a sample.

In an alternative embodiment, the device would function as a detectorfor platelet aggregation. The deterministic principle of sorting enablesa predictive outcome of the percentage of free platelets deflected inthe device. Activated platelets would form aggregates, and theaggregates would be deflected. This change can be monitored as a firstlevel sentry to alert the compromised efficacy of a platelet pack forreinfusion. The method is also generally applicable monitoring for anychange in size, e.g., through agglomeration, of particles in a sample.

Alteration

In other embodiments of the methods of this invention, cells of interestare contacted with an altering reagent that may chemically or physicallyalter the particle or the fluid in the suspension. Such applicationsinclude purification, buffer exchange, labeling (e.g.,immunohistochemical, magnetic, and histochemical labeling, cellstaining, and flow in-situ fluorescence hybridization (FISH)), cellfixation, cell stabilization, cell lysis, and cell activation.

Such methods allow for the transfer of particles from a sample into adifferent liquid. FIG. 34A shows this effect schematically for a singlestage device, FIG. 34B shows this effect for a multistage device, FIG.34C shows this effect for a duplex array, and FIG. 34D shows this effectfor a multistage duplex array. By using such methods, blood cells may beseparated from plasma. Such transfers of particles from one liquid toanother may be also employed to effect a series of alterations, e.g.,Wright staining blood on-chip. Such a series may include reacting aparticle with a first reagent and then transferring the particle to awash buffer, and then to another reagent.

FIGS. 35A, 35B, 35C illustrate a further example of alteration in atwo-stage device having two bypass channels. In this example, largeblood particles are moved from blood to buffer and collected in stage 1,medium blood particles are moved from blood to buffer in stage 2, andthen small blood particles that are not removed from the blood in stages1 and 2 are collected. FIG. 35B illustrates the size cut-off of the twostages, and FIG. 35C illustrates the size distribution of the threefractions collected.

FIG. 36 illustrates an example of alteration in a two-stage devicehaving bypass channels that are disposed between the lateral edge of thearray and the channel wall. FIG. 37 illustrates a device similar to thatin FIG. 36, except that the two stages are connected by fluidicchannels. FIG. 38 illustrates alteration in a device having two stageswith a small footprint. FIGS. 39A-39B illustrates alteration in a devicein which the output from the first and second stages is captured in asingle channel FIG. 40 illustrates another device for use in the methodsof the invention.

FIG. 41 illustrates the use of a device to perform multiple, sequentialalterations on a particle. In this method, a blood particle is movedfrom blood into a reagent that reacts with the particle, and the reactedparticle is then moved into a buffer, thereby removing the unreactedreagent or reaction byproducts. Additional steps may be added.

In another embodiment, reagents are added to the sample to selectivelyor nonselectively increase the hydrodynamic size of the particles withinthe sample. This modified sample is then pumped through an obstaclearray. Because the particles are swollen and have an increasedhydrodynamic diameter, it will be possible to use obstacle arrays withlarger and more easily manufactured gap sizes. In a preferredembodiment, the steps of swelling and size-based enrichment areperformed in an integrated fashion on a device. Suitable reagentsinclude any hypotonic solution, e.g., deionized water, 2% sugarsolution, or neat non-aqueous solvents. Other reagents include beads,e.g., magnetic or polymer, that bind selectively (e.g., throughantibodies or avidin-biotin) or non-selectively.

In an alternate embodiment, reagents are added to the sample toselectively or nonselectively decrease the hydrodynamic size of theparticles within the sample. Nonuniform decrease in particles in asample will increase the difference in hydrodynamic size betweenparticles. For example, nucleated cells are separated from enucleatedcells by hypertonically shrinking the cells. The enucleated cells canshrink to a very small particle, while the nucleated cells cannot shrinkbelow the size of the nucleus. Exemplary shrinking reagents includehypertonic solutions.

In another embodiment, affinity functionalized beads are used toincrease the volume of particles of interest relative to the otherparticles present in a sample, thereby allowing for the operation of aobstacle array with a larger and more easily manufactured gap size.

Enrichment and alteration may also be combined, e.g., where desiredcells are contacted with a lysing reagent and cellular components, e.g.,nuclei, are enriched based on size. In another example, particles may becontacted with particulate labels, e.g., magnetic beads, which bind tothe particles. Unbound particulate labels may be removed based on size.

Combination with Other Enrichment Techniques

Enrichment and alteration methods employing devices of the invention maybe combined with other particulate sample manipulation techniques. Inparticular, further enrichment or purification of a particle may bedesirable. Further enrichment may occur by any technique, includingaffinity enrichment. Suitable affinity enrichment techniques includecontact particles of interest with affinity agents bound to channelwalls or an array of obstacles.

Fluids may be driven through a device either actively or passively.Fluids may be pumped using electric field, a centrifugal field,pressure-driven fluid flow, an electro-osmotic flow, and capillaryaction. In preferred embodiments, the average direction of the fieldwill be parallel to the walls of the channel that contains the array.

Methods of Preferential Lysis

The invention further provides methods for preferentially lysing cellsof interest in a sample, e.g., to extract clinical information from acellular component, e.g., a nucleus, of the cells of interest. Ingeneral, the method employs differential lysis between the cells ofinterest and other cells (e.g., other nucleated cells) in the sample.

Lysis

Cells of interest may be lysed using any suitable method. In oneembodiment of the methods of this invention, cells may be lysed by beingcontacted with a solution that causes preferential lysis. Lysissolutions for these cells may include cell specific IgM molecules andproteins in the complement cascade to initiate complement mediatedlysis. Another kind of lysis solution may include viruses that infect aspecific cell type and cause lysis as a result of replication (see,e.g., Pawlik et al. Cancer 2002, 95:1171-81). Other lysis solutionsinclude those that disrupt the osmotic balance of cells, e.g., hypotonicor hypertonic (e.g., distilled water), to cause lysis. Other lysissolutions are known in the art. Lysis may also occur by mechanicalmeans, e.g., by passing cells through a sieve or other structure thatmechanically disrupts the cells, through the addition of heat, acoustic,or light energy to lyse the cells, or through cell-regulated processessuch as apoptosis and necrosis. Cells may also be lysed by subjectingthem to one or more cycles of freezing and thawing. Additionally,detergents may be employed to solubilize the cell membrane, lysing thecells to liberate their contents.

In one embodiment, the cells of interest are rare cells, e.g.,circulating cancer cells, fetal cells (such as fetal nucleated red bloodcells), blood cells (such as nucleated red blood cells, includingmaternal and/or fetal nucleated red blood cells), immune cells,connective tissue cells, parasites, or pathogens (such as, bacteria,protozoa, and fungi). Most circulating rare cells of interest havecompromised membrane integrity as a result of the immune attack from thehost RES (Reticulo-Endothelial-System), and accordingly are moresusceptible to lysis.

In one embodiment, the cells of interest are lysed as they flow througha microfluidic device, e.g., as described in International PublicationsWO 2004/029221 and WO 2004/113877 or as described herein. In anotherembodiment, cells of interest are first bound to obstacles in amicrofluidic device, e.g., as described in U.S. Pat. No. 5,837,115, andthen lysed. In this embodiment, the cellular components of cells ofinterest are released from the obstacles, while cellular components ofundesired cells remain bound.

Collection of Cellular Components

Desired cellular components may be separated from cell lysate by anysuitable method, e.g., based on size, weight, shape, charge,hydrophilicity/hydrophobicity, chemical reactivity or inertness, oraffinity. For example, nucleic acids, ions, proteins, and other chargedspecies may be captured by ion exchange resins or separated byelectrophoresis. Cellular components may also be separated based on sizeor weight by size exclusion chromatography, centrifugation, orfiltration. Cellular components may also be separated by affinitymechanisms (i.e., a specific binding interaction, such antibody-antigenand nucleic acid complementary interactions), e.g., affinitychromatography, binding to affinity species bound to surfaces, andaffinity-based precipitation. In particular, nucleic acids, e.g.,genomic DNA, may be separated by hybridization to sequence specificprobes, e.g., attached to beads or an array. Cellular components mayalso be collected on the basis of shape or deformability or non-specificchemical interactions, e.g., chromatography or reverse phasechromatography or precipitation with salts or other reagents, e.g.,organic solvents. Cellular components may also be collected based onchemical reactions, e.g., binding of free amines or thiols. Prior tocollection, cellular components may also be altered to enable or enhancea particular mode of collection, e.g., via denaturation, enzymaticcleavage (such as via a protease, endonuclease, exonuclease, orrestriction endonuclease), or labeling or other chemical reaction.

The level of purity required for collected cellular components willdepend on the particular manipulation employed and may be determined bythe skilled artisan. In certain embodiments, the cellular component maynot need to be isolated from the lysate, e.g., when the cellularcomponent of interest may be analyzed or otherwise manipulated withoutinterference from other cellular components. Affinity basedmanipulations (e.g., reaction with nucleic acid probes or primers,aptamers, antibodies, or sequence specific intercalating agents, with orwithout detectable labels) are amenable for use without purification ofthe cellular components.

In one embodiment, a device, e.g., as described in U.S. ApplicationPublication 2004/0144651 or as described herein, is employed to isolateparticulate cellular components of interest, e.g., nuclei, from thelysate based on size. In this embodiment, the particulate cellularcomponents of interest may be separated from other particulate cellularcomponents and intact cells using the device.

Manipulation of Cellular Components

Once released by lysis or otherwise obtained, e.g., via size basedseparation methods described herein, desired cellular components may befurther manipulated, e.g., identified, enumerated, reacted, isolated, ordestroyed. In one embodiment, the cellular components contain nucleicacid, e.g., nuclei, mitochondria, and nuclear or cytoplasmic DNA or RNA.In particular, nucleic acids may include RNA, such as mRNA or rRNA, orDNA, such as chromosomal DNA, e.g., that has been cleaved, or DNA thathas undergone apoptotic processing. Genetic analysis of the nucleic acidin the cellular component may be performed by any suitable methods,e.g., PCR, FISH, and sequencing. Genetic information may be employed todiagnose disease, status as a genetic disease carrier, or infection withpathogens or parasites. If acquired from fetal cells, geneticinformation relating to sex, paternity, mutations (e.g., cysticfibrosis), and aneuploidy (e.g., trisomy 21) may be obtained. In someembodiments, analysis of fetal cells or components thereof is used todetermine the presence or absence of a genetic abnormality, such as achromosomal, DNA, or RNA abnormality. Examples of autosomal chromosomeabnormalities include, but are not limited to, Angleman syndrome(15q11.2-q13), cri-du-chat syndrome (5p-), DiGeorge syndrome andVelo-cardiofacial syndrome (22q11.2), Miller-Dieker syndrome (17p13.3),Prader-Willi syndrome (15q11.2-q13), retinoblastoma (13q14),Smith-Magenis syndrome (17p11.2), trisomy 13, trisomy 16, trisomy 18,trisomy 21 (Down syndrome), triploidy, Williams syndrome (7q11.23), andWolf-Hirschhorn (4p-). Examples of sex chromosome abnormalities include,but are not limited to, Kallman syndrome (Xp22.3), steroid sulfatedeficiency (STS) (Xp22.3), X-linked ichthiosis (Xp22.3), Klinefeltersyndrome (XXY); fragile X syndrome; Turner syndrome; metafemales ortrisomy X; and monosomy X. Other less common chromosomal abnormalitiesthat can be analyzed by the systems herein include, but are not limitedto, deletions (small missing sections); microdeletions (a minute amountof missing material that may include only a single gene); translocations(a section of a chromosome is attached to another chromosome); andinversions (a section of chromosome is snipped out and reinserted upsidedown). In some embodiments, analysis of fetal cells or componentsthereof is used to analyze SNPs and predict a condition of the fetusbased on such SNPs. If acquired from cancer cells, genetic informationrelating to tumorgenic properties may be obtained. If acquired fromviral or bacterial cells, genetic information relating to thepathogenicity and classification may be obtained. For non-geneticcellular components, the components may be analyzed to diagnose diseaseor to monitor health. For example, proteins or metabolites from rarecells, e.g., fetal cells, may be analyzed by any suitable method,including affinity-based assays (e.g., ELISA) or other analyticaltechniques, e.g., chromatography and mass spectrometry.

General Considerations

Samples may be employed in the methods described herein with or withoutpurification, e.g., stabilization and removal of certain components.Some sample may be diluted or concentrated prior to introduction intothe device.

In another embodiment of the methods of this invention, a sample iscontacted with a microfluidic device containing a plurality ofobstacles, e.g., as described in U.S. Pat. No. 5,837,115 or as describedherein. Cells of interest bind to affinity moieties bound to theobstacles in such a device and are thereby enriched relative toundesired cells, e.g., as described in WO 2004/029221.

In another embodiment of the methods of the invention employing asimilar device, cells of non-interest bind to affinity moieties bound tothe obstacles, while allowing the cells of interest to pass throughresulting in an enriched sample with cells of interest, e.g., asdescribed in WO 2004/029221. The sized based method and theaffinity-based method may also be combined in a two-step method tofurther enrich a sample in cells of interest.

In another embodiment of the methods of the invention, a cell sample ispre-filtered by contact with a microfluidic device containing aplurality of obstacles disposed such that particles above a certain sizeare deflected to travel in a direction not parallel to the averagedirection of fluid flow, e.g., as described in U.S. ApplicationPublication 2004/0144651 or as described herein.

EXAMPLES Example 1. A Silicon Device Multiplexing 14 3-Stage ArrayDuplexes

FIGS. 42A-42E show an exemplary device of the invention, characterizedas follows.

Dimension: 90 mm×34 mm×1 mm

Array design: 3 stages, gap size=18, 12, and 8 μm for the first, secondand third stage, respectively. Bifurcation ratio=1/10. Duplex; singlebypass channel

Device design: multiplexing 14 array duplexes; flow resistors for flowstability

Device fabrication: The arrays and channels were fabricated in siliconusing standard photolithography and deep silicon reactive etchingtechniques. The etch depth is 150 μm. Through holes for fluid access aremade using KOH wet etching. The silicon substrate was sealed on theetched face to form enclosed fluidic channels using a blood compatiblepressure sensitive adhesive (9795, 3M, St Paul, Minn.).

Device Packaging: The device was mechanically mated to a plasticmanifold with external fluidic reservoirs to deliver blood and buffer tothe device and extract the generated fractions.

Device Operation: An external pressure source was used to apply apressure of 2.4 PSI to the buffer and blood reservoirs to modulatefluidic delivery and extraction from the packaged device.

Experimental conditions: human blood from consenting adult donors wascollected into K₂EDTA vacutainers (366643, Becton Dickinson, FranklinLakes, N.J.). The undiluted blood was processed using the exemplarydevice described above (FIG. 42F) at room temperature and within 9 hrsof draw. Nucleated cells from the blood were separated from enucleatedcells (red blood cells and platelets), and plasma delivered into abuffer stream of calcium and magnesium-free Dulbecco's PhosphateBuffered Saline (14190-144, Invitrogen, Carlsbad, Calif.) containing 1%Bovine Serum Albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, Mo.).

Measurement techniques: Complete blood counts were determined using aCoulter impedance hematology analyzer (COULTER® Ac⋅T Diff™, BeckmanCoulter, Fullerton, Calif.).

Performance: FIGS. 43A-43F shows typical histograms generated by thehematology analyzer from a blood sample and the waste (buffer, plasma,red blood cells, and platelets) and product (buffer and nucleated cells)fractions generated by the device. The following table shows theperformance over 5 different blood samples:

Performance Metrics Sample RBC Platelet WBC number Throughput removalremoval loss 1 4 mL/hr 100% 99% <1% 2 6 mL/hr 100% 99% <1% 3 6 mL/hr100% 99% <1% 4 6 mL/hr 100% 97% <1% 5 6 mL/hr 100% 98% <1%

Example 2. A Silicon Device Multiplexing 14 Single-Stage Array Duplexes

FIG. 44 shows an exemplary device of the invention, characterized asfollows.

Dimension: 90 mm×34 mm×1 mm

Array design: 1 stage, gap size=24 μm. Bifurcation ratio=1/60. Duplex;double bypass channel

Device design: multiplexing 14 array duplexes; flow resistors for flowstability

Device fabrication: The arrays and channels were fabricated in siliconusing standard photolithography and deep silicon reactive etchingtechniques. The etch depth is 150 μm. Through holes for fluid access aremade using KOH wet etching. The silicon substrate was sealed on theetched face to form enclosed fluidic channels using a blood compatiblepressure sensitive adhesive (9795, 3M, St Paul, Minn.).

Device Packaging: The device was mechanically mated to a plasticmanifold with external fluidic reservoirs to deliver blood and buffer tothe device and extract the generated fractions.

Device Operation: An external pressure source was used to apply apressure of 2.4 PSI to the buffer and blood reservoirs to modulatefluidic delivery and extraction from the packaged device.

Experimental conditions: human blood from consenting adult donors wascollected into K₂EDTA vacutainers (366643, Becton Dickinson, FranklinLakes, N.J.). The undiluted blood was processed using the exemplarydevice described above at room temperature and within 9 hrs of draw.Nucleated cells from the blood were separated from enucleated cells (redblood cells and platelets), and plasma delivered into a buffer stream ofcalcium and magnesium-free Dulbecco's Phosphate Buffered Saline(14190-144, Invitrogen, Carlsbad, Calif.) containing 1% Bovine SerumAlbumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, Mo.).

Measurement techniques: Complete blood counts were determined using aCoulter impedance hematology analyzer (COULTER® Ac⋅T Diff™, BeckmanCoulter, Fullerton, Calif.).

Performance: The device operated at 17 mL/hr and achieved >99% red bloodcell removal, >95% nucleated cell retention, and >98% platelet removal.

Example 3. Separation of Fetal Cord Blood

FIG. 45 shows a schematic of the device used to separate nucleated cellsfrom fetal cord blood.

Dimension: 100 mm×28 mm×1 mm

Array design: 3 stages, gap size=18, 12, and 8 μm for the first, secondand third stage, respectively. Bifurcation ratio=1/10. Duplex; singlebypass channel.

Device design: multiplexing 10 array duplexes; flow resistors for flowstability

Device fabrication: The arrays and channels were fabricated in siliconusing standard photolithography and deep silicon reactive etchingtechniques. The etch depth is 140 μm. Through holes for fluid access aremade using KOH wet etching. The silicon substrate was sealed on theetched face to form enclosed fluidic channels using a blood compatiblepressure sensitive adhesive (9795, 3M, St Paul, Minn.).

Device Packaging: The device was mechanically mated to a plasticmanifold with external fluidic reservoirs to deliver blood and buffer tothe device and extract the generated fractions.

Device Operation: An external pressure source was used to apply apressure of 2.0 PSI to the buffer and blood reservoirs to modulatefluidic delivery and extraction from the packaged device.

Experimental conditions: Human fetal cord blood was drawn into phosphatebuffered saline containing Acid Citrate Dextrose anticoagulants. Onemilliliter of blood was processed at 3 mL/hr using the device describedabove at room temperature and within 48 hrs of draw. Nucleated cellsfrom the blood were separated from enucleated cells (red blood cells andplatelets), and plasma delivered into a buffer stream of calcium andmagnesium-free Dulbecco's Phosphate Buffered Saline (14190-144,Invitrogen, Carlsbad, Calif.) containing 1% Bovine Serum Albumin (BSA)(A8412-100ML, Sigma-Aldrich, St Louis, Mo.) and 2 mM EDTA (15575-020,Invitrogen, Carlsbad, Calif.).

Measurement techniques: Cell smears of the product and waste fractions(FIG. 46A-46B) were prepared and stained with modified Wright-Giemsa(WG16, Sigma Aldrich, St. Louis, Mo.).

Performance: Fetal nucleated red blood cells were observed in theproduct fraction (FIG. 46A) and absent from the waste fraction (FIG.46B).

Example 4. Isolation of Fetal Cells from Maternal Blood

The device and process described in detail in Example 1 were used incombination with immunomagnetic affinity enrichment techniques todemonstrate the feasibility of isolating fetal cells from maternalblood.

Experimental conditions: blood from consenting maternal donors carryingmale fetuses was collected into K₂EDTA vacutainers (366643, BectonDickinson, Franklin Lakes, N.J.) immediately following electivetermination of pregnancy. The undiluted blood was processed using thedevice described in Example 1 at room temperature and within 9 hrs ofdraw. Nucleated cells from the blood were separated from enucleatedcells (red blood cells and platelets), and plasma delivered into abuffer stream of calcium and magnesium-free Dulbecco's PhosphateBuffered Saline (14190-144, Invitrogen, Carlsbad, Calif.) containing 1%Bovine Serum Albumin (BSA) (A8412-100ML, Sigma-Aldrich, St Louis, Mo.).Subsequently, the nucleated cell fraction was labeled with anti-CD71microbeads (130-046-201, Miltenyi Biotech Inc., Auburn, Calif.) andenriched using the MiniMACS™ MS column (130-042-201, Miltenyi BiotechInc., Auburn, Calif.) according to the manufacturer's specifications.Finally, the CD71-positive fraction was spotted onto glass slides.

Measurement techniques: Spotted slides were stained using fluorescencein situ hybridization (FISH) techniques according to the manufacturer'sspecifications using Vysis probes (Abbott Laboratories, Downer's Grove,Ill.). Samples were stained from the presence of X and Y chromosomes. Inone case, a sample prepared from a known Trisomy 21 pregnancy was alsostained for chromosome 21.

Performance: Isolation of fetal cells was confirmed by the reliablepresence of male cells in the CD71-positive population prepared from thenucleated cell fractions (FIG. 47). In the single abnormal case tested,the trisomy 21 pathology was also identified (FIG. 48).

The following examples show specific embodiments of devices of theinvention. The description for each device provides the number of stagesin series, the gap size for each stage, ε (Flow Angle), and the numberof channels per device (Arrays/Chip). Each device was fabricated out ofsilicon using DRIE, and each device had a thermal oxide layer.

Example 5

This device includes five stages in a single array.

-   Array Design: 5 stage, asymmetric array-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 10 μm    -   Stage 3: 12 μm    -   Stage 4: 14 μm    -   Stage 5: 16 μm-   Flow Angle: 1/10-   Arrays/Chip: 1-   Array Design: 5 stage, asymmetric array-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 10 μm    -   Stage 3: 12 μm    -   Stage 4: 14 μm    -   Stage 5: 16 μm-   Flow Angle: 1/10-   Arrays/Chip: 1

Example 6

This device includes three stages, where each stage is a duplex having abypass channel. The height of the device was 125 μm.

-   Array Design: symmetric 3 stage array with central collection    channel-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 12 μm    -   Stage 3: 18 μm    -   Stage 4:    -   Stage 5:-   Flow Angle: 1/10-   Arrays/Chip: 1-   Other: Central collection channel-   Array Design: Symmetric 3 stage array with central collection    channel-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 12 μm    -   Stage 3: 18 μm    -   Stage 4:    -   Stage 5:-   Flow Angle: 1/10-   Arrays/Chip: 1-   Other: Central collection channel

FIG. 49A shows the mask employed to fabricate the device. FIGS. 49B-49Dare enlargements of the portions of the mask that define the inlet,array, and outlet. FIGS. 50A-50G show SEMs of the actual device.

Example 7

This device includes three stages, where each stage is a duplex having abypass channel “Fins” were designed to flank the bypass channel to keepfluid from the bypass channel from re-entering the array. The chip alsoincluded on-chip flow resistors, i.e., the inlets and outlets possessedgreater fluidic resistance than the array. The height of the device was117 μm.

-   Array Design: 3 stage symmetric array-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 12 μm    -   Stage 3: 18 μm    -   Stage 4:    -   Stage 5:-   Flow Angle: 110-   Array's/Chip: 10-   Other: large fin central collection channel on-chip flow resistors-   Array Design: 3 stage symmetric array-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 12 μm    -   Stage 3: 18 μm    -   Stage 4:    -   Stage 5:-   Flow Angle: 1/10-   Arrays/Chip: 10-   Other: Large fin central collection channel on-chip flow resistors

FIG. 51A shows the mask employed to fabricate the device. FIGS. 51B-51Dare enlargements of the portions of the mask that define the inlet,array, and outlet. FIGS. 52A-52F show SEMs of the actual device.

Example 8

This device includes three stages, where each stage is a duplex having abypass channel “Fins” were designed to flank the bypass channel to keepfluid from the bypass channel from re-entering the array. The edge ofthe fin closest to the array was designed to mimic the shape of thearray. The chip also included on-chip flow resistors, i.e., the inletsand outlets possessed greater fluidic resistance than the array. Theheight of the device was 138 μm.

Array Design: 3 stage symmetric array Gap Sizes: Stage 1:  8 μm Stage 2:12 μm Stage 3: 18 μm Stage 4: Stage 5: Flow Angle: 1/10 Arrays/Chip: 10Other: alternate large fin central collection channel on-chip flowresistors

-   Array Design: 3 stage symmetric array-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 12 μm    -   Stage 3: 18 μm    -   Stage 4:    -   Stage 5:-   Flow Angle: 1/10-   Arrays/Chip: 10-   Other: Alternate large fin central collection channel on-chip flow    resistors

FIG. 45A shows the mask employed to fabricate the device. FIGS. 45B-45Dare enlargements of the portions of the mask that define the inlet,array, and outlet. FIGS. 532A-532F show SEMs of the actual device.

Example 9

This device includes three stages, where each stage is a duplex having abypass channel “Fins” were optimized using Femlab to flank the bypasschannel to keep fluid from the bypass channel from re-entering thearray. The edge of the fin closest to the array was designed to mimicthe shape of the array. The chip also included on-chip flow resistors,i.e., the inlets and outlets possessed greater fluidic resistance thanthe array. The height of the device was 139 or 142 μm.

-   Array Design: 3 stage symmetric array-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 12 μm    -   Stage 3: 18 μm    -   Stage 4:    -   Stage 5:-   Flow Angle: 1/10-   Array/Chip: 10-   Other: Femlab optimized central collection channel (Femlab I)    on-chip flow resistors-   Array Design: 3 stage symmetric array-   Gap Sizes: Stage 1: 8 μm    -   Stage 2: 12 μm    -   Stage 3: 18 μm    -   Stage 4:    -   Stage 5:-   Flow Angle: 1/10-   Arrays/Chip: 10-   Other: Femlab optimized central collection channel (Femlab 1)    on-chip flow resistors

FIG. 54A shows the mask employed to fabricate the device. FIGS. 54B-54Dare enlargements of the portions of the mask that define the inlet,array, and outlet. FIGS. 55A-55S show SEMs of the actual device.

Example 10

This device includes a single stage, duplex device having a bypasschannel disposed to receive output from the ends of both arrays. Theobstacles in this device are elliptical. The array boundary was modeledin Femlab. The chip also included on-chip flow resistors, i.e., theinlets and outlets possessed greater fluidic resistance than the array.The height of the device was 152 μm.

-   Array Design: single stage symmetric array-   Gap Sizes.: Stage 1: 24 μm    -   Stage 2:    -   Stage 3:    -   Stage 4:    -   Stage 5:    -   Flow Angle: 1/60-   Arrays/Chip: 14-   Other: central barrier    -   ellipsoid posts    -   on-chip resistors    -   Femlab modeled array boundary-   Array Design: Single stage symmetric array-   Gap Sizes: Stage 1: 24 μm    -   Stage 2:    -   Stage 3:    -   Stage 4:    -   Stage 5:-   Flow Angle: 1/60-   Arrays/Chip: 14-   Other: Central barrier    -   Ellipsoid posts    -   On-chip resistors    -   Femlab modeled array boundary

FIG. 44A shows the mask employed to fabricate the device. FIGS. 44B-44Dare enlargements of the portions of the mask that define the inlet,array, and outlet. FIGS. 56A-56C show SEMs of the actual device.

Example 11

Though the following examples focus on extraction of a purifiedpopulation of nuclei of circulating fetal cells from whole maternalblood, the methods described are generic for isolation of cellularcomponents from other cells.

Isolation of Fetal Nuclei

FIG. 57 shows a flowchart for a method of isolating fetal nuclei from amaternal blood sample. The method results in the preferential lysis ofred blood cells (FIG. 58).

Several embodiments of a method that isolates from whole blood apurified population of nuclei from circulating cells of interest forgenomic analysis are described below:

a) The method includes microfluidic processing, as described herein, ofwhole blood to 1) generate an enriched sample of nucleated cells bydepletion of 1 to 3 log of the number of enucleated red blood cells andplatelets, 2) release fetal nuclei by microfluidic processing of theenriched nucleated sample to lyse residual enucleated red cells,enucleated reticulocytes, and nucleated erythrocytes, preferentiallyover nucleated maternal white blood cells, 3) separate nuclei frommaternal nucleated white blood cells by microfluidic processing througha size based device, and 4) analyze fetal genome using commerciallyavailable gene analysis tools.

b) The method can be designed to allow Steps 1 and 2 of Embodiment 1 inone pass through a microfluidic device, followed by use of a downstreamdevice, or component of a larger device, for Step 3 (see FIGS. 59 & 60).FIG. 59 shows a schematic diagram of a microfluidic device for producingconcomitant enrichment and lysis. The device employs two regions ofobstacles that deflect larger cells from the edges of the device, wherethe sample is introduced, into a central channel containing a lysissolution (e.g., a duplex device as described herein). For maternalblood, the regions of obstacles are disposed such that maternalenucleated red blood cells and platelets remain at the edges of thedevice, while fetal nucleated red blood cells and other nucleated cellsare deflected into a central channel. Once deflected into the centralchannel, the fetal red blood cells (cells of interest) are lysed. FIG.60 shows a schematic diagram for a microfluidic device for separatingnuclei (cellular component of interest) from unlysed cells. The deviceis similar to that of FIG. 59, except the obstacles are disposed suchthat nuclei remain at the edges of the device, while larger particlesare deflected to the central channel.

c) A combination method of microfluidic based generation of fetal nucleiin maternal blood sample, followed by bulk processing techniques, suchas density gradient centrifugation to separate the fetal nuclei frommaternal cells (see FIG. 61).

d) Methods and Proof of Principle

Selective Lysis and Partitioning of Nucleated Erythrocytes.Contaminating red blood cells in donor blood samples spiked with fullterm cord blood were lysed using two methods, hypotonic and ammoniumchloride lysis. Since enucleated red cells undergo lysis in hypotonicsolution faster than nucleated cells, controlling the exposure time ofthe mixed cell population in the hypotonic solution will result in adifferential lysis of cell populations based on this time. In thismethod, the cells are sedimented to form a pellet, and the plasma abovethe pellet is aspirated. Deionized water is then added, and the pelletis mixed with the water. Fifteen seconds of exposure is sufficient tolyse >95% of the enucleated red blood cells with minimal nucleated redblood cell lysis, 15 to 30 seconds of exposure is sufficient tolyse >70% of the nucleated red blood cells but <15% of other nucleatedcells, and >30 seconds will increase the percentage of lysis of othernucleated cells. After the desired exposure time, a 10×HBSS (hypertonicbalanced salt) solution is added to return the solution back to isotonicconditions. Exposure to ammonium chloride lysing solutions at standardconcentrations (e.g., 0.15 M isotonic solution) will lyse the bulk ofred blood cells with minimal effects on nucleated cells. When theosmolality of the lysing solution is decreased to create a hypotonicammonium chloride solution, the bulk of nucleated red blood cells arelysed along with the mature red blood cells.

Density centrifugation methods were used to obtain an enrichedpopulation of lymphocytes. An aliquot of these lymphocytes were exposedto a hypotonic ammonium chloride solution for sufficient time tolyse >95% of the cells. These nuclei were then labeled with Hoechst33342 (bisbenzimide H 33342), a specific stain for AT rich regions ofdouble stranded DNA, and added back to the original lymphocytepopulation to create a 90:10 (cell: nuclei) mixture. This mixture wasfed into a device that separated cells from nuclei based on size, asdepicted in FIG. 60, and the waste and product fractions were collectedand the cell: nuclei ratio contained in each fraction were measured.

Density Gradient Centrifugation of Lysed Product. The lysed nuclei ofmixed cell suspensions that have been processed through a differentiallysis procedure can be enriched by adding a sucrose cushion solution tothe lysate. This mixture is then layered on a pure sucrose cushionsolution and then centrifuged to form an enriched nuclei pellet. Theunlysed cells and debris are aspirated from the supernatant; the nucleipellet is re-suspended in a buffer solution and then cytospun onto glassslides.

Acid Alcohol Total Cell lysis and Nuclear RNA FISH for Targeted CellIdentification. Product obtained from a device that separated cellsbased on size, as depicted in FIG. 60, was exposed to an acid alcoholsolution (methanol. acetic acid 3:1 v/v) for 30 minutes on ice resultingin the lysis of >99% of enucleated cells and >99.0% lysis of nucleatedcells. A hypotonic treatment by exposing the cells to salt solution(0.6% NaCl) for 30 minutes to swell the nuclei before acid alcohol lysiscan also be included. The released nuclei can be quantitativelydeposited onto a glass slide by cytospin and FISHed (FIG. 66). The cellsof interest, such as fetal nucleated erythrocytes, can be identifiedusing RNA-FISH with probes for positive selection, such as zeta-,epsilon, gamma-globins, and negative selection such as beta-globin oranalyzing the length of telomeres. Other methods for distinguishingbetween fetal and non-fetal cells are known in the art, e.g., U.S. Pat.No. 5,766,843.

Example 12

FIG. 62 shows a device that is optimized for separation of particles inblood. It is a one-stage device with a fixed gap width of 22 μm, with 48multiplexed arrays for parallel sample processing. The parameters of thedevice are as follows:

Array Design: L5 Gap Sizes: Stage 1: 22 μm Flow Angle: 1/50 Arrays/Chip:48 Nominal Depth 150 μm Device Footprint 32 mm × 64 mm Design FeaturesMultiplexed single arrays Optimized bypass channels Flow stabilizationFlow-feeding and Flow- extracting boundaries

Blood was obtained from pregnant volunteer donors and diluted 1:1 withDulbecco's phosphate buffered saline (without calcium andmagnesium)(iDPBS). Blood and Running Buffer (iDPBS with 1% BSA and 2 mMEDTA) were delivered using an active pressure of 0.8 PSI to the deviceengaged with a manifold as described in Example 13. Blood was separatedinto two components nucleated cells in Running Buffer and enucleatedcells and plasma proteins in Running Buffer. Both components wereanalyzed using a standard impedance counter. The component containingnucleated cells was additionally characterized using a propidium iodidestaining solution in conjunction with a standard Nageotte countingchamber to determine total nucleated cell loss. Data collected were usedto determine blood process volume (mL), blood process rate (mL/hr),RBC/platelet removal, and nucleated cell retention. The following tableprovides results of cell enrichments employing this device:

Volume 26.5   8 15.4  17  19 Processed (mL) Throughput 10.6 10.0 11.89.8 9.8 (mL/h) WBC in the  0.013%  0.012%  0.005%  0.014%  0.030% waste/input WBC (Nageotte) RBC 99.993% 99.992% 99.997% 99.995% 99.999%Removal Platelet  >99.6%  >99.7%  >99.7%  >99.7%  >99.7% Removal

Example 13

An exemplary manifold into which a microfluidic device of the inventionis inserted is shown in FIG. 63. The manifold has two halves betweenwhich a microfluidic device of the invention is disposed. One half ofthe manifold includes separate inlets for blood and buffer, each ofwhich is connected to a corresponding fluid reservoir. The channels inthe device are oriented so that they connect to the reservoirs viathrough holes in the device. Typically, the device is orientedvertically, and the processed blood is collected as it drips out of theproduct outlet. A region around the product outlet of the microfluidicdevice may also be marked with a hydrophobic substance, e.g., from apermanent marker, to limit the size of drops formed. The device alsoincludes two hydrophobic vent filters, e.g., 0.2 μm PTFE filters. Thesefilters allow air trapped in the device to be displaced by aqueoussolutions, but do not let the liquid pass at low pressures, e.g., <5psi.

To prime the device, buffer, e.g., Dulbecco's PBS with 1% bovine serumalbumin (w/v) and 2 mM EDTA, is degassed for 5-10 min under reducedpressure and while being stirred. The buffer is then pumped into thedevice via the buffer inlet in the manifold at a pressure of <5 psi. Thebuffer then fills the buffer chamber by displacing air through thehydrophobic vent filter and then fills the channels in the microfluidicdevice and the blood chamber. A hydrophobic vent filter connected to theblood chamber allows for the displacement of air in the chamber. Oncethe blood chamber is filled, buffer is pumped into the blood inlet. Incertain embodiments, after 1 minute of priming at 1 psi, the blood inletis clamped, and the pressure is increased to 3 psi for 3 minutes.

Example 14

A fetal nRBC population enriched by any of the devices described hereinis subjected to hypotonic shock by adding a large volume of low ionicstrength buffer, e.g., deionized water to lyse enucleated RBCs and nRBCsselectively and release their nuclei. The hypotonic shock is thenterminated by adding an equal volume of a high ionic strength buffer.The released nuclei, which may be subsequently harvested throughgradient centrifugation such as passage through a solution of iodixanolin water, p=1.32 g/mL, are analyzed.

FIG. 64 illustrates the selective lysis of fetal nRBCs vs. maternalnRBCs as a function of the duration of exposure to lysing conditions.This selective lysis procedure also can be used to lyse selectivelyfetal nRBCs in a population of cells composed of fetal nRBC, maternalnRBC, enucleated fetal and maternal RBCs, and fetal and maternal whiteblood cells. Using distilled water to induce hypotonic shock for a giventime period and then adding an equal volume of 10× salt solution, suchas PBS, to halt it, fetal nRBCs and maternal nRBCs were lysed over timeduring which the number of lysed (non-viable) fetal nRBCs increased by afactor of 10, whereas the number of lysed maternal nRBCs increased by asmaller multiple. At any given time point, the lysed cells were stainedwith propidium iodide and were concentrated through gradientcentrifugation to determine the ratio of lysed fetal nRBCs vs. maternalnRBCs. An optimized time duration can be determined and applied toenrich selectively for fetal nRBCs nuclei.

Example 15

To lyse enucleated RBCs and maternal nucleated RBCs selectively, asample enriched in fetal nRBCs is treated with a RBC lysis buffer, suchas 0.155 M NH₄Cl, 0.01 M KHCO₃, 2 mM EDTA, 1% BSA with a carbonicanhydrase inhibitor, such as acetazolamide (e.g., at 0.1-100 mM), toinduce lysis, followed by termination of the lysis process using a largevolume of balanced salt buffer, such as 10× volume of 1×PBS, or balancedsalt buffer, such as 1×PBS, with an ion exchange channel inhibitor suchas 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid (DIDS). Thesurviving fetal cells may then be subjected to additional rounds ofselection and analysis.

K562 cells, to simulate white blood cells, were labeled with Hoechst andcalcein AM at room temperature for 30 minutes (FIG. 65). These labeledK562 cells were added to blood specimens, followed by the addition ofbuffer (0.155 M NH₄Cl, 0.01 M KHCO₃, 2 mM EDTA, 1% BSA, and 10 mMacetazolamide) (the ratio of buffer volume to spiked blood volume is3:2). The spiked blood specimens were incubated at room temperature for4 hours with periodic gentle agitation. The fraction of viable cells ineach spiked specimen were determined by measuring the green fluorescenceat 610 nm at multiple time-points. Cell lysis is observed only afterthree minutes of treatment (in the absence of DIDS).

Example 16

A sample enriched in fetal nRBC, e.g., by any of the devices or methodsdiscussed herein, may be lysed and analyzed for genetic content.Possible methods of cell lysis and isolation of the desired cells orcell components include:

-   -   a) A sample enriched in fetal nRBC may be subjected to total        cell lysis to remove cytoplasm and isolate the nuclei. Nuclei        may be immobilized through treatment with fixing solution, such        as Carnoy's fix, and adhesion to glass slides. The fetal nuclei        may be identified by the presence of endogenous fetal targets        through immunostaining for nuclear proteins and transcription        factors or through differential hybridization, RNA FISH of fetal        pre-mRNAs (Gribnau et al. Mol Cell 2000. 377-86; Osborne et al.        Nat Gene. 2004. 1065-71; Wang et al. Proc. Natl. Acad.        Sci. 1991. 7391-7395; Alfonso-Pizarro et al. Nucleic Acids        Research. 1984. 8363-8380.) These endogenous fetal targets may        include globins such as zeta-, epsilon-, gamma-, delta-, beta-,        alpha- and non-globin targets such as I-branching enzyme (Yu et        al., Blood. 2003 101:2081), N-acetylglucosamine transferase, or        IgnT. The oligo nucleotide probes employed by RNA FISH may be        either for intron-exon boundaries or other regions, which        uniquely identify the desired target or by analyzing the length        of telomeres.    -   b) A sample enriched in fetal nRBC may be lysed selectively        using treatments with buffers and ion exchange inhibitors        described in example 15 to isolate fetal cells. The surviving        fetal cells may be further subjected to selection by the        presence or absence of intracellular markers such as globins and        I-branching beta 1, 6-N-acetylglucosaminyltransferase or surface        markers such as antigen I. In another embodiment, the enriched        fetal nRBCs can be subjected to selective lysis to remove both        the enucleated RBCs and maternal nRBCs as described in Example        15, followed by a complement mediated cell lysis using an        antibody against CD45, a surface antigen present in all        nucleated white blood cells. The resulting intact fetal nRBCs        should be free of any other contaminating cells.    -   c) A sample enriched in fetal nRBC may be lysed through        hypotonic shock as described in Example 14 to isolate fetal        nuclei. Nuclei may be immobilized through treatment with fixing        solution, such as Carnoy's fix, and adhesion to glass slides.

Once isolated, the desired cells or cell components (such as nuclei) maybe analyzed for genetic content. FISH may be used to identify defects inchromosomes 13 and 18 or other chromosomal abnormalities such as trisomy21 or XXY. Chromosomal aneuploidies may also be detected using methodssuch as comparative genome hybridization. Furthermore, the identifiedfetal cells may be examined using micro-dissection. Upon extraction, thefetal cells' nucleic acids may be subjected to one or more rounds of PCRor whole genome amplification followed by comparative genomehybridization, or short tandem repeats (STR) analysis, genetic mutationanalysis such as single nucleotide point mutations (SNP), deletions, ortranslocations.

Example 17

The product obtained from a device as depicted in FIG. 60 including 3 mlof erythrocytes in 1×PBS is treated with 50 mM sodium nitrite/0.1 mMacetazolamide for 10 minutes. The cells are then contacted with a lysisbuffer of 0.155 M NH₄Cl, 0.01 M KHCO₃, 2 mM EDTA, 1% BSA and 0.1 mMacetazolamide, and the lysis reaction is stopped by directly drippinginto a quenching solution containing BAND 3 ion exchanger channelinhibitors such as 4,4′-diisothiocyanostilbene-2,2′-disulphonic acid(DIDS). The enucleated RBCs and nucleated RBCs are counted afterWright-Giemsa staining, and FISH is used to count the fetal nRBCs.Values are then compared to an unlysed control. One such experimentalresult is shown below:

Before After Cell Lysis Lysis Recovery % eRBCs 2.6 × 10⁶ 0.03 × 10⁶ ~1%nRBCs 42 26 62% fnRBCs  6  4 68%

Example 18

Chaotropic Salt or Detergent Mediated Total Lysis and Oligo-NucleotideMediated Enrichment of Apoptotic DNA from Fetal Nucleated RBCs. Theproduct obtained from a device as depicted in FIG. 60 is lysed in achaotropic salt solution, such as buffered guanidinium hydrochloridesolution (at least 4.0 M), guanidinium thiocyanate (at least 4.0 M) or abuffered detergent solution such as tris buffered solution with SDS. Thecell lysate is then incubated at 55° C. for 20 minutes with 10 μl of 50mg/ml protease K to remove proteins and followed by a 5 minutes at 95°C. to inactive protease. The fetal nRBCs undergo apoptosis when enteringmaternal blood circulation, and this apoptotic process leads to DNAfragmentation of fetal nRBC DNA. By taking advantage of reduced size offetal nRBCs DNA and higher efficiency of isolating smaller DNA fragmentsover intact genomic DNA using oligonucleotide mediated enrichment, theapoptotic fetal nRBCs DNA can be selectively enriched throughhybridization to oligonucleotides in solution, attached to beads, orbound to an array or other surface in order to identify the uniquemolecular markers such as short tandem repeats (STR). Afterhybridization, the unwanted nucleic acids or other contaminants may bewashed away with a high salt buffered solution, such as 150 mM sodiumchloride in 10 mM Tris HCL pH 7.5, and the captured targets thenreleased into a buffered solution, such as 10 mM Tris pH 7.8, ordistilled water. The apoptotic DNA thus enriched is then analyzed usingthe methods for analysis of genetic content, e.g., as described inExample 16.

Example 19

FIG. 67 shows a flowchart detailing variations on lysis procedures thatmay be performed on maternal blood samples. Although illustrated asbeginning with Enriched Product, e.g., produced using the devices andmethods described herein, the processes may be performed on any maternalblood sample. The chart illustrates that lysis may be employed to lyse(i) wanted cells (e.g., fetal cells) selectively, (ii) wanted cells andtheir nuclei selectively, (iii) all cells, (iv) all cells and theirnuclei, (v) unwanted cells (e.g., maternal RBCs, WBCs, platelets, or acombination thereof), (vi) unwanted cells and their nuclei, and (vii)lysis of all cells and selective lysis of nuclei of unwanted cells. Thechart also shows exemplary methods for isolating released nuclei(devices and methods of the invention may also be sued for this purpose)and methods for assaying the results.

Example 20

This is an example of titrating whole cell lysis within a microfluidicenvironment. A blood sample enriched using size based separation asdescribed herein was divided into 4 equal volumes. Three of the volumeswere processed through a microfluidic device capable of transporting thecells into a first pre-defined medium for a defined path length withinthe device and then into a second pre-defined medium for collection. Thevolumetric cell suspension flow rate was varied to allow controlledincubation times with the first pre-defined medium along the definedpath length before contacting the second pre-defined medium. In thisexample DI water was used as the first pre-defined medium and 2×PBS wasused as the second predefined medium. Flow rates were adjusted to allowincubation times of 10, 20, or 30 seconds in DI water before the cellswere mixed with 2×PBS to create an isotonic solution. Total cell numbersof the 3 processed volumes and the remaining unprocessed volume werecalculated using a Hemacytometer

Starting Cell Sample Count Final Cell Count % Remaining 1 unprocessed6.6 × 10⁶ 6.6 × 10⁶  100% 2 10 second 6.6 × 10⁶ 7.2 × 10⁵ 10.9% exposure3 20 second 6.6 × 10⁶ 4.6 × 10⁵  6.9% exposure 4 30 second 6.6 × 10⁶ 3.4× 10⁵  5.2% exposure

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference. Variousmodifications and variations of the described method and system of theinvention will be apparent to those skilled in the art without departingfrom the scope and spirit of the invention. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention that are obvious to thoseskilled in the art are intended to be within the scope of the invention.

Other embodiments are in the claims.

1-168. (canceled)
 169. A method of enriching a sample in fetal cellsrelative to maternal cells, the method comprising: enriching a mixtureof maternal and fetal cells for fetal cells relative to maternal cellsto produce an enriched sample; lysing all cells in the enriched sampleto release nuclei, depositing the nuclei onto a solid support; andperforming genetic analysis of fetal DNA from the nuclei.
 170. Themethod of claim 169, wherein the enriching comprises introducing themixture of maternal and fetal cells into a device capable of enrichingfetal cells relative to maternal cells based on size, shape,deformability, or affinity
 171. The method of claim 169, wherein thesample is selected from blood, lymph, cerebrospinal fluid, urine, andcervical lavage.
 172. The method of claim 169, wherein the geneticanalysis comprises sequencing.
 173. The method of claim 169, wherein thegenetic analysis comprises polymerase chain reaction (PCR) or wholegenome amplification (WGA).
 174. The method of claim 169, wherein thegenetic analysis comprises analysis of short tandem repeats (STR). 175.The method of claim 169, wherein the genetic analysis comprises analysisof single nucleotide point mutations (SNP), deletions, ortranslocations.
 176. The method of claim 169, wherein the geneticanalysis comprises detecting chromosomal aneuploidies.
 177. The methodof claim 176, wherein the chromosomal aneuploidies are detected usingcomparative genome hybridization.
 178. The method of claim 169, whereinthe genetic analysis comprises determining the presence or absence of agenetic abnormality.
 179. The method of claim 178, wherein the geneticabnormality is a chromosomal, DNA, or RNA abnormality.
 180. The methodof claim 179, wherein the chromosomal abnormality comprises an autosomalchromosome abnormality or a sex chromosome abnormality.
 181. The methodof claim 180, wherein the chromosomal abnormality comprises an autosomalchromosome comprising Angleman syndrome (15qll.2-q 13), cri-du-chatsyndrome (5p-), Di George syndrome and Velo-cardiofacial syndrome (22ql1.2), Miller-Dieker syndrome (17p13.3), Prader-Willi syndrome(15qll.2-q13), retinoblastoma (31q14), Smith-Magenis syndrome (17pl1.2), trisomy 13, trisomy 16, trisomy 18, trisomy 21 (Down syndrome),triploidy, Williams syndrome (7 q 11.23), or Wolf-Hirschhom (4p-). 182.The method of claim 180, wherein the chromosomal abnormality comprises asex chromosome abnormality comprising Kallman syndrome (Xp22.3), steroidsulfate deficiency (STS) (Xp22.3), X-linked ichthiosis (Xp22.3),Klinefelter syndrome (XXY); fragile X syndrome; Turner syndrome;metafemales or trisomy X; or monosomy X.
 183. The method of claim 169,wherein the solid support is a glass slide.
 184. The method of claim169, wherein the lysing comprises contacting cells with a protease. 185.The method of claim 169, wherein the lysing comprises contacting cellswith a hypotonic solution.
 186. The method of claim 169, wherein thelysing comprises contacting cells with a lysis buffer.
 187. The methodof claim 169, further comprising contacting nuclei with an endonuclease.188. The method of claim 169, further comprising contacting nuclei withan exonuclease.
 189. The method of claim 169, wherein extracted fetalDNA is bound to an array or other surface.
 190. The method of claim 169,wherein the enriching comprises introducing the mixture of maternal andfetal cells into a microfluidic device comprising a channel having astructure that deterministically directs fetal nucleated cells in afirst direction and at least some maternal cells in a second directionbased on deterministic lateral displacement.
 191. The method of claim190, wherein the microfluidic device is a duplex device comprising achannel comprising a first section comprising first and second outerregions, each outer region comprising a structure that deterministicallydirects particles having a hydrodynamic size above a critical size in afirst direction and particles having a hydrodynamic size below thecritical size in a second direction, wherein the first and second outerregions are aligned in parallel in the channel.
 192. The method of claim169, wherein the genetic analysis comprises hybridizing fetal nucleicacids to oligonucleotides in solution, attached to beads, or bound to anarray or other surface to identify unique molecular markers.